LOCUS       sequence-449671-        6569 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual reporter of Ca2+ levels and AMPK activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6569)
  AUTHORS   Erdogan YC, Pilic J, Gottschalk B, Yigit EN, Zaki AG, Ozturk G, 
            Eroglu E, Okutan B, Sommer NG, Weinberg AM, Schindl R, Graier WF, 
            Malli R
  TITLE     Development of a Dual Reporter System to Simultaneously Visualize 
            Ca(2+) Signals and AMPK Activity.
  JOURNAL   ACS Sens. 2024 Aug 21. doi: 10.1021/acssensors.4c01058.
  PUBMED    39167044
REFERENCE   2  (bases 1 to 6569)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Sens. 
            2024 Aug 21. doi: 10.1021/acssensors.4c01058."
COMMENT     Sequence Label: GCaMP-AMPK-SPARK
FEATURES             Location/Qualifiers
     source          1..6569
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    151..272
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(162..181)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     216..235
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      387..433
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      463..599
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    644..715
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(941..1529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1021..1040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(2275..2343,2344..3135)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2493..2512)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      3148..3284
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    3436..3507
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(3512..3558)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    3576..3624
                     /note="synthetic polyadenylation signal"
     enhancer        3705..4008
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4010..4212
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4163..4183
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4439..4456
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(4496..4504,4505..4519)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             4547..4603
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     complement(5204..5223)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             5975..6028
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
ORIGIN
        1 cactcagaga aattgaagag ctgctccgaa agatcatcga ggacagtgta cgctcagtgg
       61 ccgagctcga ggacatcgag aaatggttga agaagatatg agctagcttg actgactgag
      121 atacagcgta ccttcagctc acagacatga taagatacat tgatgagttt ggacaaacca
      181 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
      241 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
      301 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
      361 gtattggccc atctctatcg gtatcgtagc ataacccctt ggggcctcta aacgggtctt
      421 gaggggtttt ttgtgcccct cgggccggat tgctatctac cggcattggc gcagaaaaaa
      481 atgcctgatg cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata
      541 cggcttcccc aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag
      601 gtcgtcagct atcctgcagg cgatctctcg atttcgatca agacattcct ttaatggtct
      661 tttctggaca ccactagggg tcagaagtag ttcatcaaac tttcttccct ccctaatctc
      721 attggttacc ttgggctatc gaaacttaat taaccagtca agtcagctac ttggcgagat
      781 cgacttgtct gggtttcgac tacgctcaga attgcgtcag tcaagttcga tctggtcctt
      841 gctattgcac ccgttctccg attacgagtt tcatttaaat catgtgagca aaaggccagc
      901 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
      961 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     1021 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     1081 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     1141 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     1201 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     1261 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     1321 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     1381 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     1441 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     1501 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     1561 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     1621 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     1681 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     1741 gtctatttcg ttcatccata gttgcattta aatttccgaa ctctccaagg ccctcgtcgg
     1801 aaaatcttca aacctttcgt ccgatccatc ttgcaggcta cctctcgaac gaactatcgc
     1861 aagtctcttg gccggccttg cgccttggct attgcttggc agcgcctatc gccaggtatt
     1921 actccaatcc cgaatatccg agatcgggat cacccgagag aagttcaacc tacatcctca
     1981 atcccgatct atccgagatc cgaggaatat cgaaatcggg gcgcgcctgg tgtaccgaga
     2041 acgatcctct cagtgcgagt ctcgacgatc catatcgttg cttggcagtc agccagtcgg
     2101 aatccagctt gggacccagg aagtccaatc gtcagatatt gtactcaagc ctggtcacgg
     2161 cagcgtaccg atctgtttaa acctagatat tgatagtctg atcggtcaac gtataatcga
     2221 gtcctagctt ttgcaaacat ctatcaagag acaggatcag caggaggctt tcgcatgagt
     2281 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     2341 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcgcgagtg
     2401 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     2461 cgctttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     2521 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     2581 tattcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     2641 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gattggagga
     2701 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     2761 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     2821 gcaatggcaa caaccttgcg taaactatta actggcgaac tacttactct agcttcccgg
     2881 caacagttga tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     2941 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     3001 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     3061 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     3121 attaagcatt ggtaaccgat tctaggtgca ttggcgcaga aaaaaatgcc tgatgcgacg
     3181 ctgcgcgtct tatactccca catatgccag attcagcaac ggatacggct tccccaactt
     3241 gcccacttcc atacgtgtcc tccttaccag aaatttatcc ttaagatccc gaatcgttta
     3301 aactcgactc tggctctatc gaatctccgt cgtttcgagc ttacgcgaac agccgtggcg
     3361 ctcatttgct cgtcgggcat cgaatctcgt cagctatcgt cagcttacct ttttggcagc
     3421 gatcgcggct cccgacatct tggaccatta gctccacagg tatcttcttc cctctagtgg
     3481 tcataacagc agcttcagct acctctcaat tcaaaaaacc cctcaagacc cgtttagagg
     3541 ccccaagggg ttatgctatc aatcgttgcg ttacacacac aaaaaaccaa cacacatcca
     3601 tcttcgatgg atagcgattt tattatctaa ctgctgatcg agtgtagcca gatctagtaa
     3661 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     3721 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     3781 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     3841 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     3901 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     3961 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgct gatgcggttt
     4021 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     4081 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     4141 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     4201 ataagcagag ctggtttagt gaaccgtcag atcagatctt tgtcgatcct accatccact
     4261 cgacacaccc gccagcggcc gcatgggagg aggcggctca ggaagtggag agggatcaac
     4321 taagatgcgt cgggtagcaa ctctcgtgga cctgggtacg ggcggctcag agctggacgg
     4381 tggcctggag agcggacttc gaagtggcag tggcggcggc gacattatga tgggaagtca
     4441 tcatcatcat catcatggaa tggccagtat gactggtggc cagcaaatgg ggagagatct
     4501 gtacgacgat gacgataagg atctggccac aatggtggac agttcacgcc gaaagtggaa
     4561 taagacaggc cacgcagtcc gcgcaatagg tcggctgagt agcttggaga acgtttatat
     4621 caaggccgac aagcagaaga acggcatcaa ggcgaacttc aagatccgcc acaacatcga
     4681 ggacggcgga gtgcagctcg cttaccacta ccagcagaac accccaattg gcgacgggcc
     4741 cgtgctcctt cctgacaacc actacctgtc tgtacagtca aaactgtcaa aagacccaaa
     4801 cgaaaagcga gatcacatgg tactcctcga gttcgtcaca gcagccggga tcacactcgg
     4861 tatggacgag ctgtacaagg gtgggactgg cgggagcatg gtcagtaagg gcgaggagct
     4921 gttcaccggc gtggtgccga ttctcgtcga gttagacggt gacgtcaacg gtcacaagtt
     4981 cagcgtgtca ggagagggcg agggcgatgc aacttacggt aaacttacac tcaagttcat
     5041 ctgcaccacc ggaaagctgc ccgtcccttg gcccacttta gtgacgacac tgacttacgg
     5101 cgtgcagtgc ttctctcgtt acccagacca catgaagcag catgacttct tcaagtccgc
     5161 catgcccgaa ggatacatcc aggagagaac aatcttcttc aaagacgacg gcaactacaa
     5221 gactcgagca gaggtgaagt tcgaaggcga cacactggta aaccggatcg agctgaaggg
     5281 cattgacttc aaagaggacg gtaacatact ggggcacaag ctcgagtaca accttcctga
     5341 ccaactgact gaagagcaga tcgccgaatt taaagaggaa ttcagccttt tcgacaagga
     5401 cggcgatggg accataacga ccaaggagtt gggaacggtc atgcggtctc tgggtcagaa
     5461 ccctacagaa gcagagcttc aggacatgat taatgaagtg gatgcggacg gtgacggcac
     5521 tatagacttc cctgagttcc tgacaatgat ggctcgtaaa atgaaatacc gggacacgga
     5581 agaggaaata agagaagcct tcggtgtttt cgataaggat ggcaatgggt acatatcagc
     5641 tgcagagctt aggcacgtga tgactaacct gggtgaaaag ttgaccgatg aagaggttga
     5701 tgaaatgatc agggaagccg acatcgatgg ggatggacag gtaaactacg aagagttcgt
     5761 gcaaatgatg actgcgaagg gttctggcag cgcgggtggg agtgcaggcg gatctgcagg
     5821 cggttcagct ggcgggtccg caggtggctc cgcaggcggc agcgccggtg gcagccgggg
     5881 cgaaattgct aagagcttaa aggagattgc taagagcctg aaggagatcg cctggtcttt
     5941 gaaggagatc gccaagagcc tgaagggcgg aaccgagggt agagggtcac tcctgacatg
     6001 cggcgacgtt gaagagaacc cagggcccaa gttagcaacc atgcacaagt tcagccagga
     6061 gcagattggc gagaacatcg tgtgcagggt catatgcaca acaggccaga tcccgattag
     6121 ggacctcagc gccgacatat ctcaggtgct gaaggagaaa cgctctatta agaaagtgtg
     6181 gacattcggg cggaaccccg catgcgacta ccatctgggc aacattagca ggttgtctaa
     6241 caagcacttc cagattctgc tgggcgagga cggcaacctg ttgcttaacg acatcagcac
     6301 caacggaact tggttgaacg gacaaaaggt ggaaaagaac tctaaccagt tgttgtcaca
     6361 gggcgacgag attaccgttg gggttggggt agagtcagat atcctcagct tagtcatatt
     6421 tattaacgac aagttcaagc agtgtctgga gcaaaacaag gttgaccggg gctcaggctc
     6481 tgccggcgga agtgcgggtg gctccgccgg tggatcagcc ggcggaagcg cgggcggatc
     6541 agcaggcggg agtgcgggcg gctctcgaa
//
